Treated apatite particles for ultrasound imaging

ABSTRACT

Treated apatite particles are disclosed for enhancing radical diagnostic imaging such as magnetic resonance imaging (&#34;MRI&#34;), magnetic resonance spectroscopy (&#34;MRS&#34;), magnetic resonance spectroscopy imaging (&#34;MRSI&#34;), X-ray diagnostic imaging, and ultrasound imaging. Novel coating and manufacturing techniques are disclosed to control particle size and particle aggregation resulting in compositions for organ specific imaging of the liver, spleen, gastrointestinal tract, or tissue disease states is obtained. Depending on the diagnostic imaging technique, apatite particles are treated to be paramagnetic, radiopaque, or echogenic. The apatite particles may also be fluorinated to form stable fluoroapatite compositions useful for  19  F imaging. Also disclosed are diagnostic compositions and methods of performing medical diagnostic procedures which involve administering to a warm-blooded animal a diagnostically effective amount of the abovedescribed apatite particles and then performing the medical diagnostic procedure.

This is a divisional of pending prior application Ser. No. 08/271,921filed on Jul. 6, 1994, U.S. Pat. No. 5,468,465, which is a divisionalapplication of U.S. Ser. No. 07/948,540 filed on Sep. 22, 1992 issued asU.S. Pat. No. 5,344,640; which is a continuation-in-part application ofU.S. Ser. No. 07/784,325 filed on Oct. 22, 1991 now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to treated apatite particles and their use inmedical diagnostic imaging techniques, such as magnetic resonanceimaging ("MRI"), magnetic resonance spectroscopy ("MRS"), magneticresonance spectroscopy imaging ("MRSI"), X-ray, and ultrasound. Thepresent invention also includes novel apatite particles, manufacturingmethods, and coating compositions which prevent particle aggregation,improve particle stability, and permit functionalization of the particlesurface.

The use of contrast agents in diagnostic medicine is rapidly growing. InX-ray diagnostics, for example, increased contrast of internal organs,such as the kidneys, the urinary tract, the digestive tract, thevascular system of the heart (angiography), etc., is obtained byadministering a contrast agent which is substantially radiopaque. Inconventional proton MRI diagnostics, increased contrast of internalorgans and tissues may be obtained by administering compositionscontaining paramagnetic metal species which increase the relaxivity ofsurrounding protons. In ultrasound diagnostics, improved contrast isobtained by administering compositions having acoustic impedancesdifferent than that of blood and other tissues.

Often it is desirable to image a specific organ or tissue. Effectiveorgan- or tissue-specific contrast agents accumulate in the organ ortissue of interest.

From the foregoing, it would be an important advancement in the art toprovide organ specific medical diagnostic imaging agents. Specifically,it would be an improvement in the art to provide organ specific MRI,X-ray, and ultrasound contrast agents.

Such medical diagnostic imaging agents are disclosed and claimed herein.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for improvedmedical diagnostic imaging. The imaging agents are derived from apatiteparticles including, but not limited to, hydroxyapatite (sometimesreferred to as "hydroxylapatite"), fluoroapatite, iodoapatite,carbonate-apatite, and mixtures and derivatives thereof. As used herein,the term fluoroapatite includes pure fluoroapatite as well as mixturesof fluoroapatite, hydroxyapatite, iodoapatite, and carbonate-apatite.Likewise, hydroxyapatite, iodoapatite, and carbonate-apatite areintended to include the pure and mixed forms. Since hydroxyapatite is anatural bone constituent, it is well tolerated and generally safe.

By controlling the particle size and route of administration, organspecific imaging of the liver, spleen, gastrointestinal tract, or bloodpool is obtained. Typical particle sizes are in the range from about 10nm to about 50 μm depending upon the organ or disease state to beimaged, the mechanism of delivery of the particles to the organ ordisease state, and the medical diagnostic imaging technique utilized. Inaddition, apatite particles within the range from 1 nm to about 50 nmmay also be used to image the blood pool.

Depending on the diagnostic imaging technique, apatite particles aretreated to be paramagnetic, radiopaque, or echogenic. For example,paramagnetic species may be incorporated into the apatite particles toimprove magnetic resonance contrast, and radiopaque species may beincorporated into the apatite particles to provide X-ray contrast.Particle density, and corresponding echogenic characteristics, can becontrolled to impart low or high acoustic impedance relative to blood.The apatite particles may also be fluorinated to form stable,fluoroapatite compositions useful for ¹⁹ F imaging. Incorporating aparamagnetic metal species in flouroapatite or hydroxyapatite particlesmay reduce ¹⁹ F and proton relaxivity, thereby enhancing MRI, MRS, orMRSI.

Also disclosed are diagnostic compositions and methods of performingmedical diagnostic procedures which involve administering to awarm-blooded animal a diagnostically effective amount of theabove-described apatite particles and then performing the medicaldiagnostic procedure.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for improvedmedical diagnostic imaging. As used herein, medical diagnostic imagingincludes magnetic resonance imaging ("MRI"), magnetic resonancespectroscopy ("MRS"), magnetic resonance spectroscopy imaging ("MRSI"),X-ray contrast imaging, and ultrasound imaging. The diagnostic imagingagents of the present invention are derived from apatite-like particles.

As used herein, apatite particles include apatite-like minerals of thegeneral formula Ca_(n) M_(m) X_(r) Y_(s), where M is a paramagnetic,radiopaque, or radioactive metal ion or stoichiometric mixture of metalions having a valence of 2+ or 3+, X is a simple anion, Y is atetrahedral oxyanion, carbonate, tetrahedral anion, or mixtures thereof,m is from 1-10, n is from 1-10, and r and s are adjusted as needed toprovide charge neutrality. Where M is a 2+ metal ion, then m+n=10, andwhere M is a 3+ metal ion, then m+1.5n=10.

Possible metal ions which can be used in the apatite particles of thepresent invention include: chromium(III), manganese(II), iron(II),iron(III), praseodymium(III), neodymium(III) , samarium(III) ,ytterbium(III) , gadolinium(III) , terbium(III) , dysprosium(III) ,holmium(III), erbium(III), or mixtures of these with each other or withalkali or alkaline earth metals. Typical simple anions which can be usedin the apatite particles of the present invention include: OH⁻, F⁻, Br⁻I⁻, 1/2[CO₃ ²⁻ ], or mixtures thereof. The tetrahedral oxyanions used inthe present invention may optionally include radiopaque metals orradioactive metals. Suitable tetrahedral oxyanions are non-oxidizing andstable to hydrolysis. Examples of suitable tetrahedral oxyanions for usein the present invention include: AsO₄ ³⁻, WO₄ ²⁻, MoO₄ ²⁻, VO₄ ³⁻, SiO₄⁴⁻, and GeO₄ ⁴⁻.

By controlling the particle size, organ specific imaging of the liver orgastrointestinal tract is obtained. When apatite particles having a sizein the range from about 5 nm to about 2 μm are injected into thevascular system, the particles collect in the liver or spleen (the RESsystem) because the normal function of these organs is to purify theblood of foreign particles. Once the particles have collected in theliver or spleen, these organs may be imaged by the desired medicaldiagnostic imaging technique. Apatite particles having a larger size inthe range from 200 nm to about 50 μm may be used to image thegastrointestinal ("GI") tract. Larger particles may be convenientlyadministered orally or rectally according to conventional administrationtechniques.

Depending on the diagnostic imaging technique, apatite particles aretreated to be paramagnetic, radiopaque, or echogenic. For example,paramagnetic metal species may be incorporated into the apatiteparticles to improve magnetic resonance contrast, and radiopaque speciesmay be incorporated into the apatite particles to provide X-raycontrast. Particle density, and corresponding echogenic characteristics,can be controlled to impart low or high acoustic impedance relative toblood. The apatite particles may also be fluorinated to form stable,nontoxic fluoroapatite compositions useful for ¹⁹ F imaging. Thepresence of a paramagnetic metal species in fluoroapatite orhydroxyapatite particles may reduce ¹⁹ F and proton relaxivity, therebyenhancing MRI, MRS, or MRSI.

Preparation of Apatite Particles

Methods for preparing hydroxyapatite, having the formula Ca₁₀ (OH)₂(PO₄)₆, are well known in the art. Apatites in which the OH⁻ is replacedwith simple anions, including F⁻, Br⁻, I⁻, or 1/2[CO₃ ²⁻ ], may beprepared by modifying the process for preparing hydroxyapatite. Apatitederivatives in which calcium is replaced by metal ions, such asparamagnetic, radiopaque, or radioactive metal ions, may also beprepared and used within the scope of the present invention. Usefulapatites may also be prepared by replacing phosphate with oxyanions ortetrahedral anions containing radiopaque or radioactive metal species.

Stoichiometric pure hydroxyapatite has a Ca:P ratio of 1.67:1. The majorimpurity found in hydroxyapatite is tricalcium phosphate, Ca₃ (PO₄)₂,known as "TCP". This impurity can be detected by deviation from the1.67:1 Ca:P ratio (for large amounts of impurity) or by X-raydiffraction for impurity levels down to 1 percent.

Stoichiometrichydroxyapatite is prepared by adding an ammonium phosphatesolution to a solution of calcium/ ammonium hydroxide. To minimize theamount of TCP formed, it is important to have excess calcium throughoutthe addition process.

Apatite Particles for MRI Applications

The technique of MRI encompasses the detection of certain atomic nuclei(those possessing magnetic dipole moments) utilizing magnetic fields andradio-frequency radiation. It is similar in some respects to X-raycomputed tomography ("CT") in providing a cross-sectional display of thebody organ anatomy with excellent resolution of soft tissue detail. Thetechnique of MRI advantageously avoids the use of ionizing radiation.

The hydrogen atom, having a nucleus consisting of a single unpairedproton, has the strongest magnetic dipole moment of any nucleus. Sincehydrogen occurs in both water and lipids, it is abundant in the humanbody. Therefore, MRI is most commonly used to produce images based uponthe distribution density of protons and/or the relaxation times ofprotons in organs and tissues. Other nuclei having a net magnetic dipolemoment also exhibit a nuclear magnetic resonance phenomenon which may beused in magnetic resonance applications. Such nuclei include carbon-13(six protons and seven neutrons), fluorine-19 (9 protons and 10neutrons), sodium-23 (11 protons and 12 neutrons), and phosphorus-31 (15protons and 16 neutrons).

In an MRI experiment, the nuclei under study in a sample (e.g. protons,¹⁹ F, etc.) are irradiated with the appropriate radio-frequency ("RF")energy in a controlled gradient magnetic field. These nuclei, as theyrelax, subsequently emit RF energy at a sharp resonance frequency. Theresonance frequency of the nuclei depends on the applied magnetic field.

According to known principles, nuclei with appropriate spin when placedin an applied magnetic field (B, expressed generally in units of gaussor Tesla (10⁴ gauss)) align in the direction of the field. In the caseof protons, these nuclei precess at a frequency, F, of 42.6 MHz at afield strength of 1 Tesla. At this frequency, an RF pulse of radiationwill excite the nuclei and can be considered to tip the netmagnetization out of the field direction, the extend of this rotationbeing determined by the pulse, duration and energy. After the RF pulse,the nuclei "relax" or return to equilibrium with the magnetic field,emitting radiation at the resonant frequency. The decay of the emittedradiation is characterized by two relaxation times, T₁ and T₂. T₁ is thespin-lattice relaxation time or longitudinal relaxation time, that is,the time taken by the nuclei to return to equilibrium along thedirection of the externally applied magnetic field. T₂ is the spin-spinrelaxation time associated with the dephasing of the initially coherentprecession of individual proton spins. These relaxation times have beenestablished for various fluids, organs, and tissues in different speciesof mammals.

For protons and other suitable nuclei, the relaxation times Thd 1 and T₂are influenced by the environment of the nuclei (e.g., viscosity,temperature, and the like). These two relaxation phenomena areessentially mechanisms whereby the initially imparted radio-frequencyenergy is dissipated to the surrounding environment. The rate of thisenergy loss or relaxation can be influenced by certain other nuclei ormolecules (such as nitroxide radicals) which are paramagnetic. Chemicalcompounds incorporating paramagnetic nuclei or molecules maysubstantially alter the T₁ and T₂ values for nearby nuclei having amagnetic dipole moment. The extent of the paramagnetic effect of thegiven chemical compound is a function of the environment within which itfinds itself.

In general, paramagnetic ions of elements with an atomic number of 21 to29, 42 to 44 and 58 to 70 have been found effective as MRI contrastingagents. Examples of suitable paramagnetic ions include chromium(III),manganese(II), iron(II), iron(III), cobalt(II), nickel(II), copper(II),praseodymium(III), neodymium(III), samarium(III), gadolinium(III),dysprosium III), and ytterbium(III). Certain molecules, such asnitroxide radicals, also exhibit paramagnetic properties.

Paramagnetic metal ions may be incorporated into the apatite structureby replacement of calcium sites. Apatite doping in the range from about1% to 100% is possible, depending upon the particular metal species. Inmost cases, apatite doping with metal ions in the range from about 1% to25% is expected. Currently, the preferred metals from a toxicity andefficacy viewpoint are iron and manganese. With iron dopedhydroxyapatite particles, any iron released from metabolized orsolubilized particles would join the body's pool of iron, with calciumand phosphate also going to their respective body pools. Manganese ispreferred because of its higher relaxivity properties and affinity forliver tissue. Moreover, the liver has a clearance mechanism formanganese, thereby reducing residual toxicity.

Metal doped hydroxyapatite is prepared by mixing a basic (pH 12)phosphate solution with a calcium/paramagnetic metal solution at nativepH. Alternatively, the calcium/paramagnetic metal solution could bebasic (pH 12) if the solution also contains a ligand to preventhydrolysis of the paramagnetic metal. The ligand could either be left inthe hydroxyapatite matrix or "ashed out" by sintering the hydroxyapatitebetween 200° C. and 1100° C. Any strong chelating ligands may be used,such as polyamino polycarboxylic acid derivatives which are well knownin the art.

It has been found that the paramagnetic ions incorporated into theapatite particle tend to oxidize during particle synthesis. To preventmetal oxidation, manufacturing techniques have been developed tominimize the amount of oxygen in the aqueous reactant solutions. Forexample, two such manufacturing techniques are (1) synthesis at hightemperature, such as 100° C. and (2) degassing the aqueous reactantsolutions with an inert gas such as argon, nitrogen, or helium. Anunexpected benefit of these techniques is the ability to prepare smallerparticles, in the range from 50 nm to about 1 μm.

Antioxidants, such as gentisic acid and ascorbic acid, added duringapatite particle synthesis may also be used to prevent metal ionoxidation. Reducing agents, such as NaBH₄, have been found to reducemetal ions that are unintentionally oxidized during apatite particlesynthesis.

Paramagnetic apatite particles may also be prepared by adsorbingparamagnetic metal ions onto the particle surface. For example,manganese can be surface-adsorbed to hydroxyapatite particles by takinga slurry of hydroxyapatite, adding Mn(NO₃)₂ and applying energy, such asultrasonic power or heat, to the resulting mixture. The resultingmixture can be separated by either centrifugation and decantation or byfiltration. The resulting solid is washed with large amounts of water toremove excess manganese. The same procedure may be used with otherparamagnetic cations. The amount of manganese adsorbed onto the particlesurface, as a percentage of the total calcium in the particle, is in therange from about 0.1% to about 10%. Such particles exhibit very highrelaxivities and rapid liver enhancement in magnetic resonance imagingstudies.

Paramagnetic metal species may also be adsorbed onto apatite particlesurfaces through the use of bifunctional coating agents. Examples ofpossible bifunctional coating agents are chelating agents having one ormore phosphonate groups capable of adsorption to the apatite particlesurface. One currently preferred bifunctional coating agent is thefunctionalized polyphosphonatediethylenetriaminepenta(methylenephosphonic acid), abbreviated DETAPMDP,having the following structure: ##STR1##

Once adsorbed to the apatite particle surface, the bifunctional coatingagent may form complexes with paramagnetic metal ions. These particlesalso exhibit very high relaxivities and rapid liver enhancement inmagnetic resonance imaging studies.

In some cases, the concentration of nuclei to be measured is notsufficiently high to produce a detectable MR signal. For instance, since¹⁹ F is present in the body in very low concentration, a fluorine sourcemust be administered to a subject to obtain a measurable MR signal.Signal sensitivity is improved by administering higher concentrations offluorine or by coupling the fluorine to a suitable "probe" which willconcentrate in the body tissues of interest. High fluorine concentrationmust be balanced against increased tissue toxicity. It is also currentlybelieved that a fluorine agent should desirably contain magneticallyequivalent fluorine atoms in order to obtain a clear, strong signal.

Fluoroapatites, useful as ¹⁹ F imaging agents, are prepared by replacingthe OH⁻ with stoichiometric or nonstoichiometric quantities of F⁻.Fluoroapatites may also be synthesized with organic phosphate estersusing the procedures described by M. Okazaki, "FluoridatedHydroxyapatites Synthesized With Organic Phosphate Ester," Biomaterials,Vol. 12, pp. 46-49, (1991). It is currently believed that all of thefluorine atoms in fluoroapatite are chemically and magneticallyequivalent. Since fluoroapatite has a high molar content of identicalfluorine atoms, it may be advantageously used as a low concentration ¹⁹F MRI agent. Fluoroapatite may also be doped with paramagnetic metalspecies, as described above, to reduce ¹⁹ F and proton relaxivity,thereby enhancing MRI, MRS, or MRSI.

Apatite Particles for X-ray Contrast Applications

The apatite particles described herein may also be adapted for deliveryof radiopaque species into the body for X-ray contrast. Typicalradiopaque species which may be incorporated into the apatite particlesinclude heavy metals, iodine, or iodinated XRCM.

Iodoapatites are prepared by replacing the OH⁻ of hydroxyapatite withstoichiometric or non-stoichiometric quantities of I⁻. Because iodine issubstantially radiopaque, iodoapatites may be used as X-ray contrastmedia ("XRCM"). By controlling the particle size, iodoapatite particlesmay be used to image the liver or spleen of the RES or thegastrointestinal tract.

Commercially available XRCM, such as Ioversol, may be incorporated inapatite particles during particle precipitation. In the case ofhydroxyapatite particles, the XRCM may be included in either thephosphate or calcium solution. The XRCM is preferably in sufficientlyhigh concentration that upon precipitation of the apatite particles, theXRCM has a concentration in the particles in the range from about 1% toabout 25%, by weight.

Certain radiopaque heavy metals, such as bismuth, tungsten, tantalum,hafnium, lanthanum and the lanthanides, barium, molybdenum, niobium,zirconium, and strontium may also be incorporated into apatite particlesto provide X-ray contrast. The radiopaque metals are incorporated intoapatite particles in the same manner as paramagnetic metal ions,described above.

Apatite Particles for Ultrasound Applications

Ultrasound is a medical diagnostic technique in which sound waves arereflected differently against different types of tissue, depending uponthe acoustic impedance of these tissues. There is interest in being ableto use some type of contrast agent to obtain an amplification ofspecific organs. Hydroxyapatite particles may be made echogenic byeither of two mechanisms: (1) reflection off high density hydroxyapatiteparticles or (2) reflection off air trapped within low densityhydroxyapatite particles.

Since hydroxyapatite is a porous material, small pockets of gas withinthe particles render them echogenic, with an impedance less than blood.An ultrasound contrast media would be provided in a two-vial kit form:one vial containing dry hydroxyapatite and the other vial containing adiluent.

For example, appropriately sized particles would be synthesized using avolatile organic solvent and then dried by freeze-drying orlyophilization. The resulting dried particles would have pores filledwith gas. Just prior to use, a second vial containing a specific volumeof a sterile aqueous diluent, such as isotonic saline and/or buffer, canbe aspirated and added to the vial of the dried hydroxyapatite. Theslurry is then mixed and immediately injected. Enough gas remains in thepores to provide echogenicity in vivo for ultrasound contrast.

Alternatively, carbonate can be incorporated into thehydroxyapatitematrix. Appropriately sized particles would be dried asdescribed above. The diluent vial would contain a weak biocompatibleacid such as, but not limited to, acetic acid, citric acid, NaH₂ PO₄,etc. The diluent and hydroxyapatite particles are mixed to allow theacid to react with the carbonate and form carbon dioxide in the particlepores according to equation A, below. The mixture would then be injectedin vivo and ultrasound imaging of the desired vasculature would proceed.

    CO.sub.3.sup.2- +2H.sup.+ →CO.sub.2 +H.sub.2 O      (A)

Echogenic hydroxyapatite particles with an impedance higher than bloodmay also be prepared. Prolonged heating or sintering at hightemperatures can render hydroxyapatite, with or without additives, intoa hardened, less porous material that is denser than blood. Densehydroxyapatite particles can transmit sound faster than blood, therebyproviding an echogenic material having an impedance higher than blood.

High impedance ultrasound contrast media may be provided as either apre-mixed single vial formulation or a two-vial kit form. Appropriatelysized particles formed and heated at optimum conditions would ultimatelybe formulated with a biocompatible aqueous diluent such as, but notlimited to, isotonic saline and/or a buffer.

Although the foregoing discussion has focused on the use of treatedhydroxyapatite particles for ultrasound contrast, it will be appreciatedthat other apatite particles may also be treated and used as ultrasoundcontrast agents.

Controlling the Particle Size and Aggregation

Various techniques are available to control the apatite particle size.For example, slower mixing rates (introduction of the precipitatinganion or cation), larger solution volumes, higher reaction temperatures,and lower concentrations generally result in smaller particles. Inaddition, sonication during precipitation, turbulent flow or impingementmixers, homogenization, and pH modification may be used to controlparticle size.

Procedures for preparing monodispersed colloidal particles that areknown in the art may be adapted for preparing submicron apatiteparticles. E. Matijevic, "Production of Monodispersed ColloidalParticles," Annual Review of Material Science, volume 15, pages 483-516,1985, which is incorporated herein by reference, describes methods forcontrolling the release of precipitating anions and cations. Forexample, when urea, CO(NH₂)₂, is heated, hydroxide ions are slowlyliberated which can cause precipitation of hydroxyapatite as submicronparticles. Likewise, precipitating cations can be released slowly bydecomposition of metal complexes, such as organometallic compounds.

In addition to chemical means for controlling the release ofprecipitating ions, mechanical means, such as computer controlledautoburets, peristaltic pumps, and syringes, may also be used to controlthe release of precipitating ions. Commercially available autoburets arecapable of releasing solutions at rates as low as 10 μL/minute. In thefuture as computer controlled equipment improves, it is expected thateven slower release rates may be obtained.

Due to the small size and nature of apatite particles, they tend toaggregate. Particle aggregation may be reduced by coating the particles.Although the reasons apatite particles aggregate is not fullyunderstood, it has been found that several different coating agents areable to inhibit particle aggregation. For example, apatite particles maybe stabilized by treatment with coating agents such as di- andpolyphosphonate-containing compounds, such as hydroxyethyldiphosphonate(HEDP), pyrophosphate, aminophosphonates; carboxylates andpolycarboxylate-containing compounds such as oxaltes and citrates;alcohols and polyalcohol-containing compounds; phosphates andpolyphosphate-containing compounds; sulfates and sulfate-containingcompounds; sulfonates and sulfonate-containing compounds; andbiomolecules such as peptides, proteins, antibodies, and lipids. Suchcoating agents stabilize the small apatite particles by reducing furtherparticle growth and promoting particle suspension.

Stabilized apatite particles are desirable for in vivo use as medicaldiagnostic imaging agents. Apatite particle can also be stabilized byaddition of small amounts of calcium sequestering anions, such ascitrate and oxalate. Such anions, which coordinate calcium, mayeffectively stabilize small apatite particles.

When used in magnetic resonance imaging, particle relaxivity is enhancedby allowing more water accessible to the particle surface. By limitingparticle size and increasing the available surface area, improvedrelaxivity is observed.

In addition to the coating agents identified above, conventionalparticle coating techniques may also be used in the manufacturingprocesses of the present invention. Typical coating techniques areidentified in International Publication Numbers WO 85/02772, WO91/02811, and European Publication Number EP 0343934, which areincorporated by reference.

For instance, agglomerated particles may be disrupted by mechanical orchemical means and then coated with polymers such as carbohydrates,proteins, and synthetic polymers. Dextran having a molecular weight inthe range from about 10,000 to about 40,000 is one currently preferredcoating material. Albumin and surfactants, such as tween 80, have alsobeen used to reduce particle aggregation. One common characteristic ofuseful apatite coating agents is their ability to modify the particlesurface charge, or zeta potential.

The currently preferred mechanical means for disrupting or subdividingagglomerated particles is sonication, but other means such as heating,other forms of particle energization, such as irradiation, and chemicalmeans, such as pH modification or combinations of these types oftreatment, such as pH modification combined with sonication may be used.

Functionalized Apatite Particles

Apatite particles may be prepared with coating agents containingreactive functional groups such as amine, active ester, alcohol, andcarboxylate. Such functional groups may be used to couple apatiteparticles to paramagnetic metal chelates, to organ or tissue specificpeptides or proteins, and to antibodies. An example of one possiblecoating agent having a reactive functional group is the following HEDPderivative: ##STR2##

Those skilled in the art will appreciate that other coating agents,modified to contain various reactive functional groups, may be used inthe present invention.

Diagnostic Pharmaceutical Formulations

The apatite particles of this invention are preferably formulated intodiagnostic compositions for enteral or parenteral administration. Forexample, parenteral formulations advantageously contain a sterileaqueous solution or suspension of treated apatite particles according tothis invention. Various techniques for preparing suitable pharmaceuticalsolutions and suspensions are known in the art. Such solutions also maycontain pharmaceutically acceptable buffers and, optionally,electrolytes such as sodium chloride. Parenteral compositions may beinjected directly or mixed with a large volume parenteral compositionfor systemic administration.

Formulations for enteral administration may vary widely, as iswell-known in the art. In general, such formulations include adiagnostically effective amount of the apatite particles in aqueoussolution or suspension. Such enteral compositions may optionally includebuffers, surfactants, thixotropic agents, and the like. Compositions fororal administration may also contain flavoring agents and otheringredients for enhancing their organoleptic qualities.

The diagnostic compositions of this invention are used in a conventionalmanner in magnetic resonance, X-ray, and ultrasound procedures. Thediagnostic compositions are administered in a sufficient amount toprovide adequate visualization, to a warm-blooded animal eithersystemically or locally to an organ or tissues to be imaged, then theanimal is subjected to the medical diagnostic procedure. Such doses mayvary widely, depending upon the diagnostic technique employed as well asthe organ to be imaged.

The following examples are offered to further illustrate the presentinvention. These examples are intended to be purely exemplary and shouldnot be viewed as a limitation on any claimed embodiment.

EXAMPLE 1 Preparation of Hydroxyapatite

A calcium nitrate solution was prepared by adding 1.18 g Ca(NO₃)₂ ·4H₂ Oto 20 mL deionized water such that the final [Ca²⁺ ]=0.25 M. The calciumnitrate solution pH was adjusted to a pH of 11 with ammonium hydroxide.An ammonium phosphate solution was prepared by adding 0.396 g (NH₄)₂HPO₄ to 5 mL of deionized water. The pH of the ammonium phosphatesolution was adjusted to a pH of 11 with ammonium hydroxide. Theammonium phosphate solution was injected into the calcium nitratesolution and vigorously stirred. The resulting precipitated particleswere examined under a microscope and estimated to have particle sizesgreater than 10 μm.

EXAMPLE 2 Preparation of Hydroxyapatite

Hydroxyapatite particles were prepared according to the procedure ofExample 1, except that the pH of the calcium nitrate solution was notadjusted to pH 11. The ammonium phosphate solution was injected into thecalcium nitrate solution and vigorously stirred. The resultingprecipitated particles were examined under a microscope and estimated tohave particle sizes greater than 10 μm.

EXAMPLE 3 Preparation of Hydroxyapatite

A calcium nitrate solution was prepared by adding 0.68 g Ca(NO)₃)₂ ·4H₂O to 5 mL deionized water such that the [Ca²⁺ ]=0.58M. The calciumnitrate solution pH was adjusted to a pH of 11 with ammonium hydroxide.An ammonium phosphate solution was prepared by adding 0.22 g (NH₄)₂ HPO₄to 10 mL of deionized water such that the [HPO₄ ²⁻ ]=0.17M. The pH ofthe ammonium phosphate solution was adjusted to 11 with ammoniumhydroxide. The ammonium phosphate solution was dripped into a vigorouslystirred calcium nitrate solution over 30 minutes. After mixing, thefinal [Ca²⁺ ]=0.19M. The resulting precipitated particles were examinedunder a microscope and estimated to have particle sizes of approximately1 μm.

EXAMPLE 4 Preparation of Hydroxyapatite Doped with a Paramagnetic MetalIon

A metal ion solution was prepared by adding 1.18 g Ca(NO₃)₂ ·4H₂ O and0.202 g Fe(NO₃)₃ ·9H₂ O to 20 mL deionized water. An ammonium phosphatesolution was prepared by adding 0.396 g (NH₄)₂ HPO₄ to 5 mL of deionizedwater. The pH of the ammonium phosphate solution was adjusted to 11 withammonium hydroxide. The ammonium phosphate solution was injected intothe metal ion solution and vigorously stirred. The resultingprecipitated particles were examined and found to have particle sizesgreater than 10 μm.

Example 5 Preparation of Fluoroapatite

Fluoroapatite is prepared by mixing 5 mL of a 0.58M solution of calciumfluoride with 10 mL of a 0.17M ammonium phosphate solution at native pH.The calcium fluoride solution is dripped into a vigorously stirredammonium phosphate solution over 30 minutes. The resulting precipitatedparticles are examined under a microscope and estimated to have particlesizes of approximately 1 μm.

EXAMPLE 6 Preparation of Fluoroapatite

Fluoroapatite is prepared by mixing 5 mL of a 0.58 M solution of calciumnitrate with 10 mL of solution containing 0.17M ammonium phosphate and0.17M ammonium fluoride. The calcium nitrate solution is dripped into avigorously stirred ammonium phosphate and ammonium fluoride solutionover 30 minutes. The resulting precipitated particles are examined undera microscope and estimated to have particle sizes of approximately 1 μm.

EXAMPLE 7 Preparation of Fluoroapatite Doped with a Paramagnetic MetalIon

Fluoroapatite doped with a paramagnetic metal ion is prepared accordingto the procedure of Example 5, except that the calcium fluoride solutionalso contains 0.058 M manganese nitrate. The calcium fluoride/manganesenitrate solution is dripped into a vigorously stirred ammonium phosphatesolution over 30 minutes. The resulting precipitated particles areexamined under a microscope and estimated to have particle sizes ofapproximately 1 μm.

EXAMPLE 8 Preparation of Iodoapatite

Iodoapatite is prepared by mixing 5 mL of a 0.58M solution of calciumiodide with 10 mL of a 0.17M ammonium phosphate solution at native pH.The calcium iodide solution is dripped into a vigorously stirredammonium phosphate solution over 30 minutes. The resulting precipitatedparticles are examined under a microscope and estimated to have particlesizes of approximately 1 μm.

EXAMPLE 9 Preparation of Iodoapatite

Iodoapatite is prepared by mixing 5 mL of a 0.58M solution of calciumnitrate with 10 mL of solution containing 0.17M ammonium phosphate and0.17M ammonium iodide. The calcium nitrate solution is dripped into avigorously stirred ammonium phosphate and ammonium iodide solution over30 minutes. The resulting precipitated particles are examined under amicroscope and estimated to have particle sizes of approximately 1 μm.

EXAMPLE 10 Preparation of Hydroxyapatite Doped with an XRCM

Hydroxyapatite particles doped with iothalamate meglumine, an ionicXRCM, are prepared according to the procedure of Example 3, except thatthe calcium nitrate solution also contains 0.058M iothalamate megluminesalt. Iothalamate has the following structure: ##STR3##

The ammonium phosphate solution is dripped using an autoburet into avigorously stirring solution of calcium nitrate and iothalamatemeglumine over 30 minutes. The resulting precipitated particles areexamined under a microscope and estimated to have submicron particlesizes.

EXAMPLE 11 Preparation of Hydroxyapatite Doped with a Radiopaque HeavyMetal

Hydroxyapatite particles doped with tungsten are prepared according tothe procedure of Example 3, except that the ammonium phosphate solutionalso contains 0.116M sodium tungstate, Na₂ WO₄. The ammoniumphosphateand sodium tungstate solution is dripped using an autoburet into avigorously stirred solution of calcium nitrate over 30 minutes. Theresulting precipitated particles are examined under a microscope andestimated to have submicron particle sizes.

EXAMPLE 12 Preparation of Hydroxyapatite Doped with Carbonate

Carbonate-doped hydroxyapatite particles are prepared according to theprocedure of Example 3, except that calcium carbonate was used insteadof calcium nitrate. The ammonium phosphate solution is dripped using acomputer controlled autoburet into a vigorously stirred calciumcarbonate solution over 30 minutes. The resulting precipitated particleswere examined under a microscope and estimated to have submicronparticle sizes.

EXAMPLE 13 Preparation of High Density Hydroxyapatite

Hydroxyapatite particles are prepared according to the procedure ofExample 3 and sintered at a temperature in the range from about 200° C.to about 1100° C. to harden and densify the particles. The denseparticles can then be mixed with a suitable pharmaceutical carrier andadministered as a high acoustic impedance ultrasound contrast media.

EXAMPLE 14 Preparation at 100° C. of Hydroxyapatite

An ammonium phosphate solution was prepared by dissolving 10.56 grams(NH₄)₂ HPO₄ in 200 mL of D.I. water. To this was added 100 mL ofconcentrated NH₄ OH with stirring. A white precipitate formed which wasdissolved by addition of 150 mL of H₂ O. This solution was stirred for 3hours at room temperature and then added dropwise (over 2 hours) via aperistaltic pump (Masterflex) to a 1000 mL three-neck round bottom flaskfitted with a dry ice/ isopropanol condenser on top of a standardwater-jacketed condenser containing a solution of 31.5 grams Ca(NO₃)₂·4H₂ O in 500 mL of H₂ O in boiling water stirred rapidly with amechanical stirrer. Reflux was continued for two hours after additionwas complete and the mixture was allowed to cool to room temperaturewith stirring overnight. The reaction mixture was centrifuged at 2300rpm and the nearly-clear supernatant discarded. The resulting white,pelleted solid was slurried with water and completely broken up by meansof a vortex mixer. The mixture was again centrifuged and the cloudysupernatant collected. The washing was repeated two separate times. Allthree washings were saved as was the remaining solid in the centrifugetubes. The calcium/phosphorous ratio and particle size of the washedparticles is summarized below:

    ______________________________________                                               Ca/P Ratio  Particle size (std. dev.)                                  ______________________________________                                        wash 1   1.65          663 (456) nm                                           wash 2   1.67          351 nm, 1853 nm.sup.†                           wash 3   1.67          190 nm, 1069 nm.sup.†                           ______________________________________                                         .sup.† Bimodal distribution noted, no standard deviations given.  

EXAMPLE 15 Preparation at 100° C. of Hydroxyapatite Doped with Mn(II)

This material was prepared according to the procedure of Example 14except that a Mn(II) (as Mn(NO₃)₂ ·H₂ O) was substituted mole-for-molefor Ca. For example, to synthesize 5% Mn incorporated into HA:

10.56 grams (NH₄)₂ HPO₄ was dissolved in 200 mL of D.I. water. To thiswas added 100 mL of concentrated NH₄ OH with stirring. A whiteprecipitate formed which was dissolved by addition of 150mL of H₂ O.This solution was stirred for 3 hours at room temperature and then addeddropwise (over 2 hours) via a peristaltic pump (Masterflex) to a 1000 mLthree-neck round bottom flask fitted with a dry ice/ isopropanolcondenser on top of a standard water-jacketed condenser containing asolution of 1.27 grams Mn(NO3)₂ ·H₂ O and 29.9 grams Ca(NO₃)₂ ·4H₂ O in500 mL of H₂ O in boiling water stirred rapidly with a mechanicalstirrer. Reflux was continued for two hours after addition was completeand the mixture was allowed to cool to room temperature with stirringovernight. The reaction mixture was centrifuged at 2300 rpm and thenearly-clear supernatant discarded. The resulting off-white, pelletedsolid was slurried with water and completely broken up by means of avortex mixer. The mixture was again centrifuged and the cloudysupernatant collected. The washing procedure was repeated two times. Allthree washings were saved as was the remaining solid in the centrifugetubes. The particle size of the particles in the supernatant increasedand the percentage of particles in the supernatant decreased (i.e., lesscloudy supernatant). Solids from supernatants could be concentrated byfurther centrifugation at 7000 rpm. The average particle size was 449 nmwith a standard deviation of 171 nm.

EXAMPLE 19 Preparation at 100° C. of Hydroxyapatite particles Doped withMn and treated with HEDP

Manganese containing hydroxyapatite particles were prepared by thefollowing general procedure (Mn/Ca mole ratios of <0.33 can be used):

A solution containing 6.5 g of (NH₄)₂ HPO₄ in 120 mL of deionized waterwas treated with 60 mL of concentrated ammonium hydroxide, NH₄ OHfollowed by 90 mL of D.I. water. The resulting mixture was stirred atroom temperature for 3 hours.

Into a 1L 3-neck round bottom flask equipped with a water cooled/lowtemperature condenser sequence (dry ice/ isopropanol bath), mechanicalstirrer and rubber septum were placed 18.3 g of Ca(NO₃)₂ ·4H₂ O and 0.7g of Mn(NO₃)₂ ·XH₂ O in 468 mL of D.I. water (Ca/Mn mole ratio=19/1,Ca+Mn=0.081 moles). The resulting solution was heated to reflux. Thephosphate/hydroxide mixture was then added dropwise over approximatelyone hour with a peristaltic addition pump. The reaction mixture wascooled to room temperature and stirred overnight. The solution was thentreated with 0.54 M HEDP (pH 6.6, 1-1.2 Ca/HEDP mole ratio) and stirredat room temperature for one hour.

The reaction mixture was then divided among six 50 mL plastic centrifugetubes and centrifuged for 15 minutes at 2400 rpm. The procedure wasrepeated with the remainder of the reaction mixture. The almost clearsupernatant was discarded and the solid in each tube resuspended to 50mL of volume with D.I. water and re-centrifuged. The milky wash was setaside and the solid washed twice more. The three washes were combinedand then centrifuged at 7000 rpm for 30 minutes. The particles remainedpelleted and the clear supernatant was decanted. The solid wasresuspended in water and re-centrifuged three more times at 7000 rpmdiscarding the supernatant after each washing. After the centrifugeworkup the solid particles were resuspended in 20-30 mL of D.I. waterand then subjected to routine analysis.

Characterization of the particle suspension gave the following results:

size (average diameter, nm): 258

relaxivity (mMolar⁻¹ sec.sup.⁻¹): 3.05

[Mn] (mole/liter): 0.11

[Ca] (mole/liter): 3.29

% Mn (mole % relative to Ca): 3.35

In magnetic resonance imaging studies, a 45% enhancement of the liverwas observed 4 hours post injection at a dose of 10 μmoles Mn/Kg animalbody weight.

EXAMPLE 17 Preparation at room temperature of Hydroxyapatite particlesDoped with Mn and treated with HEDP

Manganese containing hydroxyapatite particles were prepared by thefollowing general procedure. A procedure is described for particlescontaining 10% Mn but other percentages are also applicable.

Into a 1L erlenmeyer flask were placed 10.5 g of (NH₄)₂ HPO₄, 100 mL ofconcentrated NH₄ OH and 350 mL of D.I. water. The mixture was stirredfor two hours with a continuous heavy argon flow (degassing). In aseparate 1L erlenmeyer flask were placed 28.9 g of Ca(NO₃)₂ ·4H₂ O and2.4 g of Mn(NO₃)₂ ·XH₂ O in 400 mL of D.I. water. The metal nitratesolution was degassed with argon for 2 hours. The phosphate solution wasthen added dropwise to the rapidly stirred metal nitrate mixture overtwo hours with a peristaltic pump. A continuous argon flow wasmaintained throughout the course of the reaction. The reaction mixturewas stirred for an additional two hours after the addition was complete.

A solution of 8.3 g of a 60% solution HEDP (acid form) in 25 mL of Do I.water was degassed for 30 minutes then added in one aliquot to thehydroxyapatite mixture. The resulting slurry was stirred for 15 minutes.The entire reaction mixture was centrifuged at one time at 2400 rpm for15 minutes. The supernatant was discarded and the solid residue in eachtube resuspended in water. The slurry was re-centrifuged at 2400 rpm andthe milky supernatant was collected. The solid was resuspended twicemore and centrifuged at 2400 rpm. The three washes were combined andcentrifuged at 7000 rpm for 30 minutes. The solid pellet waswashed/centrifuged three times and the supernatants discarded. Afterwashing, the solid pellet was suspended in 30 mL of D.I. H₂ O.

Characterization of the particulate suspension produced the followingresults:

size (average diameter, nm): 229

relaxivity (mMolar-1 sec-1 ): 29.4

[Mn] (mole/liter): 0.027

[Ca] (mole/liter): 0.377

% Mn (mole % relative to Ca): 6.71

In magnetic resonance imaging studies, a 45% enhancement of the liverwas observed immediately post injection at a dose of 10 μmoles Mn/Kganimal body weight.

EXAMPLE 18 Preparation at room temperature of Hydroxyapatite Doped with10% Mn(II), Modified by Surface-Adsorbed Mn(II) and HEDP Addition

An ammonium phosphate solution was prepared by dissolving 5.3 grams(NH₄)₂ HPO₄ in 175 mL of D.I. water. To this was added 50 mL ofconcentrated NH₄ OH with stirring. This solution was degassed for 2hours (argon bubbling) with stirring and then added dropwise (over 2hours) via a peristaltic pump (Masterflex) to a solution of 1.27 gramsMn(NO₃)₂ ·H₂ O and 14.5 grams Ca(NO₃)₂ ·4H₂ O in 200 mL of H₂ O that hadalso been deaerated for 2 hours with argon as it was stirred rapidlywith a mechanical stirrer. Argon bubbling was continued during theaddition. The reaction mixture was stirred for an additional 2 hours asthe Ar bubbling continued. 1.27 g Mn(NO₃)₂ ·H₂ O in 25 mL of deaeratedH₂ O was added in one portion to the reaction slurry, followed, after 15minutes, by 4.3 grams of a 60% HEDP solution in water dissolved in 10 mLof deaerated H₂ O. The reaction mixture was centrifuged at 2300 rpm andthe nearly-clear supernatant discarded. The resulting white, pelletedsolid was slurried with water and completely broken up by means of avortex mixer. The mixture was again centrifuged and the cloudysupernatant collected. The washing was repeated two separate times. Allthree washings were saved as was the remaining solid in the centrifugetubes.

In magnetic resonance imaging studies, a 30% enhancement of the liverwas observed immediately post injection at a dose of 10 μmoles Mn/Kganimal body weight.

EXAMPLE 19 Preparation at 100° C. of Hydroxyapatite Particles Modifiedby Surface-Adsorbed Mn(II) and HEDP Addition

Into a 250 mL erlenmeyer flask were placed 6.3 g of (NH₄)₂ HPO₄ in 120mL of D.I. water. Concentrated NH₄ OH (60 mL) was added to the mixturefollowed by 90 mL of D.I. water. The solution was stirred at roomtemperature for four hours.

Into a 1L 3-neck round bottom flask equipped with a water cooled and lowtemperature condenser sequence (dry ice/isopropanol), mechanicalstirrer, and rubber septum were placed 19.0 g of Ca(NO₃)₂ ·4H₂ O in 468mL of D.I. H₂ O. The mixture was heated to reflux and thephosphate/ammonium hydroxide solution added dropwise with a peristalticpump and rapid stirring over one hour. The heating was removed when theaddition was complete. The reaction mixture was cooled to roomtemperature then stirred overnight.

The pH of the hydroxyapatite slurry was adjusted from 9.50 to 8.70 with80 mL of 0.5 N HCl. 2.1 g of Mn(NO₃)₂ ·XH₂ O in 5 mL of H₂ O was addedto the hydroxyapatite mixture and stirred for four hours. The reactionmixture became light brown in color. A solution of HEDP (0.54 M, Ca/HEDPmole ratio=1.1) was added and the resulting reaction mixture stirred atroom temperature for 3 hours. The color of the slurry becamepurple/brown.

The reaction mixture was divided among six 50 mL plastic centrifugetubes and centrifuged for 15 minutes at 2400 rpm. The supernatant wasdeep purple and clear. The solid residue was washed/centrifuged threetimes with 50mL volumes of water per tube and the three washes combined.The combined washes were centrifuged at 7000 rpm for 20 minutes. Thesolid pellets were washed/centrifuged three additional times discardingthe supernatant after each centrifuge run. The white solid residue wassuspended in 15 mL of D.I. H₂ O then subjected to routine analyses.

The analyses of the manganese adsorbed hydroxylapatite slurry gave thefollowing results:

size (average diameter, nm): 259

relaxivity (mMolar⁻¹ sec⁻¹): 13.8

[Mn] (mole/liter): 0.010

[Ca ] (mole/liter): 1.60

% Mn (mole % relative to Ca): 0.66

EXAMPLE 20 Preparation at Room Temperature of Hydroxyapatite Doped with10% Mn(II), Modified by Sequential Addition of Mn(II) and HEDP WithWashings Between Steps

The general procedure is the same as in Example 18. Before addition ofthe additional Mn(NO₃)₂, however, the reaction mixture was pH adjustedfrom 9.8 to a lower pH (7.5-9.5) and the mixture then centrifuged, theresulting solid washed with D.I. water, the Mn(NO₃)₂ added with stirringunder argon bubbling, the resultant mixture centrifuged and the solidwashed with water. In the final step the HEDP was added to the slurriedsolid and then the excess washed away with the supernatant duringcentrifugation.

In the preparation where the pH was adjusted to 9.5, 5.3 grams (NH₄)₂HPO₄ was dissolved in 175 mL of D.I. water. To this was added 50 mL ofconcentrated NH₄ OH with stirring. This solution was degassed for 2hours (argon bubbling) with stirring and then added dropwise (over 2hours) via a peristaltic pump (Masterflex) to a solution of 1.27 gramsMn(NO₃)₂ H₂ O and 14.5 grams Ca(NO₃)₂ ·4H₂ O in 200 mL of H₂ O that hadalso been deaerated for 2 hours with argon as it was stirred rapidlywith a mechanical stirrer. Argon bubbling was continued during theaddition. The reaction mixture was stirred for an additional 2 hours asthe argon bubbling continued. The pH of the reaction mixture wasadjusted from 9.8 to 9.0 with 3 N HCl with rapid stirring and argonbubbling.

1.27 g Mn(NO₃)₂ ·H₂ O in 25 mL of deaerated H₂ O was added in oneportion to the reaction slurry, followed, after 60 minutes, bycentrifugation and one washing of the resultant solid (via vortex mixingand recentrifugation). The solid was suspended in water and treated with4.3 grams of a 60% HEDP solution in water dissolved in 10 mL ofdeaerated H₂ O. After 15 minutes the reaction mixture was centrifuged at2300 rpm and the nearly-clear supernatant discarded. The resultingwhite, pelleted solid was slurried with water and completely broken upby means of a vortex mixer. The mixture was again centrifuged and thecloudy supernatant collected. The washing was repeated two separatetimes. All three washings were combined and the solids from thosewashings pelleted by centrifugation at 7000 rpm. The resulting pelletwas washed with water 3 times by suspension followed by centrifugationat 7000 rpm. The particles were analyzed and found to have an averageparticle size of 251 nm and a relaxivity, R₁ =25 mM⁻¹ sec⁻¹.

EXAMPLE 21 Preparation at 100° C. of Hydroxyapatite Particles Modifiedby Surface-Adsorbed Mn, Purified, then Treated with HEDP

Calcium hydroxyapatite particles were prepared by the followingprocedure:

A solution containing 6.5 g of (NH₄)₂ HPO₄ in 120 mL of D.I. water wastreated with 60 mL of concentrated NH₄ OH followed by 90 mL of D.I.water. The resulting solution was stirred for 3 hours at roomtemperature.

Into a 3-neck 1L round bottom flask equipped with a water cooled and lowtemperature condenser sequence (dry ice/isopropanol), mechanical stirrerand rubber septum were placed 19.4 g of Ca(NO₃)₂ ·4H₂ O in 468 mL ofD.I. water. The solution was heated to reflux. The phosphate mixture wasadded to the rapidly stirred calcium nitrate solution dropwise with aperistaltic pump over one hour. The heat was removed when the additionwas complete and the reaction mixture cooled to room temperature. Thehydroxylapatite slurry was stirred overnight at room temperature.

The pH of the reaction mixture was decreased from 9.53 to 8.50 with 169ml of 1N HCl. Manganese nitrate, Mn(NO₃)₂ ·H₂ O (2.10 g) was added tothe hydroxyapatite mixture and stirred for 1 hour and 15 minutes. Thecolor of the slurry became pale tan. The mixture was then centrifuged at2400 rpm for 15 minutes. The clear colorless supernatant was discardedand the solid washed/centrifuged with 3-50 mL aliquots of water at 2400rpm for 15 minutes per run. Half of the solid residue was suspended in200 mL of D.I. water and stirred vigorously then placed in an ultrasonicbath for 10 minutes to break apart any large clumps. The solid slurrywas then treated with 0.54 M HEDP (Ca/HEDP mole ratio=1.2) and stirredfor 1.5 hours. The color of the mixture became pale pink/purple. Theremaining half of the solid hydroxyapatite pellet was suspended in 200mL of D.I. H₂ O and set aside for characterization and analyses.

The HEDP treated hydroxyapatite fraction was divided among six 50 mLplastic centrifuge tubes and centrifuged for 15 minutes at 2400 rpm. Thesupernatant was deep purple and slightly cloudy. The solid residue wassuspended in H₂ O and centrifuged at 7000 rpm for 30 minutes. Thesupernatant was discarded and the solid pellet washed/centrifuged threemore times at 7000 rpm. The purified hydroxyapatite was suspended inapproximately 30 mL of D.I. water then characterized. The results of theanalyses are listed below.

    ______________________________________                                                         HEDP treated                                                                             untreated                                         ______________________________________                                        size (average diameter, nm)                                                                      216          34,100                                        relaxivity (mMolar.sup.-1  sec.sup.-1)                                                           38.3         0.78                                          [Mn] (mole/liter)  0.0025       0.016                                         [Ca] (mole/liter)  0.170        0.638                                         % Mn (mole % relative to Ca)                                                                     1.44         2.45                                          ______________________________________                                    

In magnetic resonance imaging studies, a 25% enhancement of the liverwas observed immediately post injection at a dose of 10 μmoles Mn/Kganimal body weight.

EXAMPLE 22 Preparation of Mn-Doped Hydroxyapatite Particles Having aFunctionalized Coating Agent

This example describes the general preparation of hydroxyapatiteparticles having a functionalized coating agent. The particles areprepared by adding 0.1-100 mole % of an appropriate coating agent to aslurry of Mn(II) substituted hydroxyapatite with 0.1-100 mole % Mn basedon the Ca used in the reaction. The mixture is stirred from 1 to 360minutes at temperatures in the range from 4° C. to 100° C. and the solidseparated from the supernatant by centrifugation. The resulting solid iscollected or subjected to repeated washings with water to remove excessions and coating agent. The solid, after resuspension in water, may betreated with a metal salt (0.01-10 mole % based on Ca in thepreparation). This is especially appropriate if the coating agentcontains a pendant chelating group to capture and hold tightly the metal(when subjected to in vitro and/or in vivo solutions). The resultantsolid is separated by centrifugation and washed 3 times with water toremove loosely attached coating agent or free metal/coating agentcomplex.

EXAMPLE 23 Preparation of Hydroxyapatite Particles treated withDiethylenetriamine-penta(methylenephosphonic acid) Followed by SurfaceAdsorption of Mn

Calcium hydroxyapatite was prepared by the following procedure thentreated with the functionalized polyphosphonate,diethylenetriamine-penta(methylenephosphonic acid), abbreviated DETAPMDPand having the following structure: ##STR4##

A basic ammonium phosphate solution was prepared using 6.34 g of (NH₄)₂HPO₄ in 120 mL of D.I. water. Concentrated ammonium hydroxide (60 mL)was added followed by 90 ml of D.I. water. The mixture was stirred for 4hours at room temperature.

A solution of 19.0 g of Ca(NO₃)₂ ·4H₂ O in 468 mL of D.I. water wasplaced in a 3-neck 1L round bottom flask. The reaction setup included amechanical stirrer, water cooled and low temperature (dryice/isopropanol) condenser arrangement, and a rubber septum. Thesolution was heated to reflux with rapid stirring. The basic phosphatesolution was added dropwise with a peristaltic pump over one hour. Theheat was removed after the addition was complete and the reactionmixture stirred overnight at room temperature.

The hydroxyapatite slurry was treated with a solution of DETAPMDP(Ca/DETAPMDP mole ratio=1.1, pH of DETAPMDP 6.3) and stirred at roomtemperature for 2.5 hours. The phosphonate treated mixture was thenreacted with Mn(NO₃)₂ ·XH₂ O (Ca/Mn mole ratio=2.3) and stirred for anadditional 3.5 hours.

The reaction mixture was divided among six 50 mL plastic centrifugetubes and centrifuged at 2400 rpm for 15 minutes. The clear supernatantwas discarded and the solid residue suspended in 50 mL of D.I. per tubeand centrifuged at 2400 rpm. The milky suspension was decanted and setaside. The solid was washed/centrifuged twice more and the three washescombined. The milky suspension was re-centrifuged at 7000 rpm for 30minutes. The clear supernatant was discarded and the solid pelletresuspended and centrifuged three additional times at 7000 rpm. Thepurified pellet was then suspended in 15 mL of D.I. water and analyzed.The following results were obtained.

size (average diameter, nm): 258

relaxivity (mMolar⁻¹ sec⁻¹): 20.3

[Mn] (mole/liter): 0.0013

[Ca] (mole/liter): 1.921

% Mn (mole % relative to Ca): 0.07

In magnetic resonance imaging studies, a 30% enhancement of the liverwas observed immediately post injection at a dose of 10 μmoles Mn/Kganimal body weight.

EXAMPLE 24 Replacement of Phosphate with Arsenate in Preparation ofHydroxyapatite and Substituted Hydroxyapatites

The procedure according to Example 17 is used except that 0.1-100 mole %arsenate is substituted for the phosphate. For example, 9.51 grams(NH₄)₂ HPO₄ and 1.49 grams Na₂ AsO₄ were dissolved in 400 mL of D.I.water. To this was added 100 mL of concentrated NH₄ OH with stirring.The rest of the procedure follows directly from Example 17.

EXAMPLE 25 Replacement of Phosphate with Vanadate in Preparation ofHydroxyapatite and Substituted Hydroxyapatites

The procedure according to Example 17 is used except that 0.1-100 molepercent vanadate is substituted for the phosphate. For example, 9.51grams (NH₄)₂ HPO₄ and 1.40 grams Na₃ VO₄ were dissolved in 400 mL ofD.I. water. To this was added 100 mL of concentrated NH₄ OH withstirring. The rest of the procedure follows directly from Example 17.

EXAMPLE 26 Preparation at 100° C. of Mn-Doped Fluoroapatite Particles

Manganese fluoroapatite was prepared by the following general procedure.Into a 5-neck 1L round bottom flask equipped with a mechanical stirrer,water cooled reflux condenser, adapter for pH electrode, and two rubbersepta for addition of reagents were placed 10.3 g of Mn(OAC)₂ ·4H₂ O in200 mL of D.I. water. The solution was degassed with heavy argonbubbling for 30 minutes. A solution of ammonium fluoride, NH₄ F (0.3 g)in 50 mL of D.I. water was prepared in a 125 mL erlenmeyer flask anddegassed for 30 minutes with argon. Into a 250 mL erlenmeyer flask wasplaced 3.3 g of (NH₄)₂ HPO₄ in 150 mL of D.I. water and degassed for 30minutes before addition.

The manganese acetate solution was heated to reflux with rapid stirring(pH 6.6) and the NH₄ F and (NH₄)₂ HPO₄ solutions were added dropwisesimultaneously with a peristaltic pump over 35 minutes. The solidprecipitated among immediate addition of reagents and was pale pink incolor. The pH of the reaction mixture dropped to 4.7 by the end of thereaction. The heating was stopped when the addition was complete. Thereaction mixture was stirred at room temperature overnight.

The apatite slurry was divided among four 50 mL plastic centrifuge tubesand centrifuged for 30 minutes at 2400 rpm. The clear supernatant wasdiscarded, and the pale pink solid was resuspended and centrifuged for30 minutes at 2400 rpm. The solid was washed and centrifuged twice moreand the clear supernatants discarded. The purified solid pellet wassuspended in 20 mL of D.I. water.

From the foregoing, it will be appreciated that the present inventionprovides organ specific medical diagnostic imaging agents for use inMRI, X-ray, and ultrasound.

The invention may be embodied in other specific forms without departingfrom its spirit or essential characteristics. The described embodimentsare to be considered in all respects only as illustrative and notrestrictive. The scope of the invention is, therefore, indicated by theappended claims rather than by the foregoing description. All changeswhich come within the meaning and range of equivalency of the claims areto be embraced within their scope.

What is claimed is:
 1. A method for enhancing ultrasound images of bodyorgans and tissues comprising:(a) administering to a patient, adiagnostically effective amount of echogenic apatite particles, in apharmaceutically acceptable carrier; and (b) imaging the organs andtissues using ultrasound techniques.
 2. A method for enhancingultrasound images of body organs and tissues as defined in claim 1,wherein the echogenic apatite particles include gas-filled pores.
 3. Amethod for enhancing ultrasound images of body organs and tissues asdefined in claim 1, wherein the echogenic apatite particles are preparedby mixing apatite particles, having a carbonate salt incorporatedtherein, with a weak biocompatible acid, such that carbon dioxide isformed within pores of the apatite particles.
 4. A method for enhancingultrasound images of body organs and tissues as defined in claim 1,wherein the echogenic apatite particles are dense particlessubstantially without pores.
 5. A diagnostic composition for enhancingultrasound contrast suitable for enteral or parenteral administration toa patient, which comprises:a diagnostically effective amount ofechogenic apatite particles having a particle size in the range fromabout 5 nm to about 50 μm, said apatite particles having a generalformula Ca₁₀ X₂ (PO₄)₆, wherein X is OH, F, Br, I, 1/2CO₃ ; and apharmaceutically acceptable carrier.
 6. A diagnostic composition asdefined in claim 5, wherein the echogenic apatite particles includegas-filled pores.
 7. A diagnostic composition as defined in claim 5,wherein the echogenic apatite particles are prepared by mixing apatiteparticles, having a carbonate salt incorporated therein, with a weakbiocompatible acid, such that carbon dioxide is formed within pores ofthe apatite particles.
 8. A diagnostic composition as defined in claim5, wherein the echogenic apatite particles are dense particlessubstantially without pores.